scholarly journals Characterization of calcium-dependent membrane binding proteins of brain cortex

1985 ◽  
Vol 229 (3) ◽  
pp. 587-593 ◽  
Author(s):  
A R Rhoads ◽  
M Lulla ◽  
P B Moore ◽  
C E Jackson
Keyword(s):  
Brain Cortex ◽  
Peptide Mapping ◽  
Bovine Brain ◽  
Particulate Fraction ◽  
Structural Homology ◽  
One Dimensional ◽  
Calcium Dependent ◽  
Stokes Radius ◽  
The Brain

Proteins of Mr 68 000, 34 000 and 32 000 were selectively extracted by EGTA from brain cortex. The three proteins that were extracted along with calmodulin were acidic, monomeric, and did not exhibit structural homology, as demonstrated by one-dimensional peptide mapping. The Mr-68 000 protein was purified to homogeneity and had a Stokes radius of 3.54 nm and S20,W value of 5.1S. Purified calmodulin, Mr-68 000 protein and two proteins of Mr 34 000 and Mr 32 000, interacted with the brain particulate fraction, with half-maximal binding occurring at 3.5 microM, 8.3 microM and 150 microM-Ca2+ respectively. Proteins were bound independently of each other and calmodulin. Pretreatment of the particulate fraction with trypsin prevented the Ca2+-dependent binding of calmodulin; however, the binding of the Mr-68 000 protein or the Mr−32 000 and −34 000 proteins was unaffected. The Mr-68 000 protein of bovine brain did not cross-react immunologically with Mr-67 000 calcimedin from chicken gizzard.

Characterization of an 80-kDa phosphoprotein involved in parietal cell stimulation

1989 ◽  
Vol 256 (6) ◽  
pp. G1070-G1081 ◽  
Author(s):  
T. Urushidani ◽  
D. K. Hanzel ◽  
J. G. Forte
Keyword(s):  
Membrane Protein ◽  
Parietal Cell ◽  
Apical Membrane ◽  
Peptide Mapping ◽  
Integral Membrane Protein ◽  
Gastric Glands ◽  
Low Speed ◽  
Calcium Dependent ◽  
Apical Membranes

When isolated rabbit gastric glands were stimulated with histamine plus isobutylmethylxanthine, a redistribution of H+-K+-ATPase, from microsomes to a low-speed pellet, occurred in association with the phosphorylation of an 80-kDa protein (80K) in the apical membrane-rich fraction purified from the low-speed pellet. Histamine alone or dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP), but not carbachol, also stimulated both the redistribution of H+-K+-ATPase and phosphorylation of 80K. Under stimulated conditions, 80K copurified in the apical membrane fraction along with H+-K+-ATPase and actin; whereas purified microsomes from resting stomach were highly enriched in H+-K+-ATPase but contained neither 80K nor actin. Treatment of the apical membranes with detergents, salts, sonication, and so on, led us to conclude that 80K is a membrane protein, unlike actin; however, the mode of association of 80K with membrane differed from H+-K+-ATPase, an integral membrane protein. Isoelectric focusing and peptide mapping revealed that 80K consists of six isomers of slightly differing pI, with 32P occurring only in the three most acidic isomers and exclusively on serine residues. Moreover, stimulation elicited a shift in the amount of 80K isomers, from basic to acidic, as well as phosphorylation. We conclude that 80K is an apical membrane protein in the parietal cell and an important substrate for cAMP-dependent, but not calcium-dependent, pathway of acid secretion.

scholarly journals Tissue-Specificity of Antibodies Raised Against TrkB and p75NTR Receptors; Implications for Platelets as Models of Neurodegenerative Diseases

2021 ◽  
Vol 12 ◽  
Author(s):  
Samuel Fleury ◽  
Imane Boukhatem ◽  
Jessica Le Blanc ◽  
Mélanie Welman ◽  
Marie Lordkipanidzé
Keyword(s):  
Central Nervous System ◽  
Neurological Disorders ◽  
Blood Circulation ◽  
Tissue Specificity ◽  
Receptor Kinase ◽  
Calcium Dependent ◽  
Neuronal Growth Factor ◽  
The Central Nervous System ◽  
The Brain

Platelets and neurons share many similarities including comparable secretory granule types with homologous calcium-dependent secretory mechanisms as well as internalization, sequestration and secretion of many neurotransmitters. Thus, platelets present a high potential to be used as peripheral biomarkers to reflect neuronal pathologies. The brain-derived neurotrophic factor (BDNF) acts as a neuronal growth factor involved in learning and memory through the binding of two receptors, the tropomyosin receptor kinase B (TrkB) and the 75 kDa pan-neurotrophic receptor (p75NTR). In addition to its expression in the central nervous system, BDNF is found in much greater quantities in blood circulation, where it is largely stored within platelets. Levels 100- to 1,000-fold those of neurons make platelets the most important peripheral reservoir of BDNF. This led us to hypothesize that platelets would express canonical BDNF receptors, i.e., TrkB and p75NTR, and that the receptors on platelets would bear significant resemblance to the ones found in the brain. However, herein we report discrepancies regarding detection of these receptors using antibody-based assays, with antibodies displaying important tissue-specificity. The currently available antibodies raised against TrkB and p75NTR should therefore be used with caution to study platelets as models for neurological disorders. Rigorous characterization of antibodies and bioassays appears critical to understand the interplay between platelet and neuronal biology of BDNF.

scholarly journals Purification and characterization of the 27,000 Da calcium-binding protein of bovine brain

1987 ◽  
Vol 244 (2) ◽  
pp. 401-408 ◽  
Author(s):  
M Tokuda ◽  
N C Khanna ◽  
D M Waisman
Keyword(s):  
Calcium Binding ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sedimentation Coefficient ◽  
Bovine Brain ◽  
Stokes Radius ◽  
Acidic Nature ◽  
Bovine Pancreas

A Ca2+-binding protein named CAB-27 was purified from bovine brain 100,000 g supernatant. The protein has a molecular mass of 27,000 Da as determined by SDS/polyacrylamide-gel electrophoresis and 35,500 Da by sedimentation-coefficient and Stokes-radius analysis. The protein contains about 26% Glx and Asx and 13% basic residues. The acidic nature of the molecule is confirmed by its pI of 4.80. In the presence of 3 mM-MgCl2 and 150 mM-KCl, CAB-27 binds 2.0 mol of Ca2+/mol of protein, with an apparent Kd of 0.2 microM. Ca2+-binding is unaffected by prior incubation of the protein at 80 degrees C for 2 min. Brain contains about 130 mg of CAB-27/kg. Immunoblotting identified CAB-27 in several bovine tissues; it appears to be particularly rich in brain and kidney. In addition, CAB-27 is identified as an inhibitor of bovine pancreas phospholipase A2 in vitro. The inhibitory activity of CAB-27 was 20-fold less potent than lipocortin. On the basis of the Ca2+-binding properties, intracellular concentration and tissue distribution of this protein, we suggest that CAB-27 may be an important intracellular Ca2+ receptor.

scholarly journals Characterization of calmodulin-dependent cyclic nucleotide phosphodiesterase isoenzymes

1994 ◽  
Vol 299 (1) ◽  
pp. 97-100 ◽  
Author(s):  
R K Sharma ◽  
J Kalra
Keyword(s):  
Cyclic Nucleotide ◽  
Catalytic Properties ◽  
Bovine Brain ◽  
Fine Tuning ◽  
Kinetic Properties ◽  
Complex Interactions ◽  
Second Messenger Systems ◽  
The Brain ◽  
Phosphodiesterase Isoenzymes

Calmodulin-dependent phosphodiesterase (CaMPDE) is one of the key enzymes involved in the complex interactions which occur between the cyclic-nucleotide and Ca2+ second-messenger systems. Calmodulin-dependent phosphodiesterase exists in different isoenzymic forms, which exhibit distinct molecular and/or catalytic properties. The kinetic properties suggest that the 63 kDa brain isoenzyme is distinct from the brain 60 kDa and heart and lung CaMPDE isoenzymes. The CaMPDE isoenzymes of 60 kDa from brain, heart and lung are regulated by calmodulin, but the affinities for calmodulin are different. At identical concentrations of calmodulin, the bovine heart CaMPDE isoenzyme is stimulated at a much lower Ca2+ concentration than the bovine brain or lung isoenzymes. The bovine lung CaMPDE isoenzyme contains calmodulin as a tightly bound subunit, so that a change in calmodulin concentration had no effect on the [Ca2+]-dependence of activation of this isoenzyme. These observations are consistent with the notion that differential regulation by calmodulin and Ca2+ is an important function of these isoenzymes, which provide fine-tuning mechanisms for calmodulin action.

scholarly journals Specific localization of scallop gill epithelial calmodulin in cilia

1982 ◽  
Vol 92 (3) ◽  
pp. 622-628 ◽  
Author(s):  
EW Stommel ◽  
RE Stephens ◽  
HR Masure ◽  
JF Head
Keyword(s):  
Matrix Protein ◽  
Peptide Mapping ◽  
Electrophoretic Analysis ◽  
Bovine Brain ◽  
Affinity Column ◽  
Calcium Dependent ◽  
Calcium Buffer ◽  
Membrane Matrix ◽  
Ciliary Protein ◽  
Cell Fractions

Calmodulin has been isolated and characterized from the gill of the bay scallop aequipecten irradians. Quantitative electrophoretic analysis of epithelial cell fractions show most of the calmodulin to be localized in the cilia, specifically in the detergent- solubilized membrane-matrix fraction. Calmodulin represents 2.2 +/- 0.3 percent of the membrane-matrix protein or 0.41 +/- 0.5 percent of the total ciliary protein. Its concentration is at least 10(-4) M if distributed uniformly within the matrix. Extraction in the presence of calcium suggests that the calmodulin is not bound to the axoneme proper. The ciliary protein is identified as a calmodulin on the basis of its calcium- dependent binding to a fluphenazine-sepharose affinity column and its comigration with bovine brain calmodulin on alkaline-urea and SDS polyacrylamide gels in both the presence and absence of calcium. Scallop ciliary calmodulin activates bovine brain phosphodiesterase to the same extent as bovine brain and chicken gizzard calmodulins. Containing trimethyllysine and lacking cysteine and tryptophan, the amino acid composition of gill calmodulin is typical of known calmodulins, except that it is relatively high in serine and low in methionine. Its composition is less acidic than other calmodulins, in agreement with an observed isoelectric point approximately 0.2 units higher than that of bovine brain. Comparative tryptic peptide mapping of scallop gill ciliary and bovine brain calmodulins indicates coincidence of over 75 percent of the major peptides, but at least two major peptides in each show no near-equivalency. Preliminary results using ATP-reactivated gill cell models show no effect of calcium at micromolar levels on ciliary beat or directionality of the lateral cilia, the cilia which constitute the vast majority of those isolated. However, ciliary arrest will occur at calcium levels more than 150 muM. Because calmodulin usually functions in the micromolar range, its role in this system is unclear. Scallop gill ciliary calmodulin may be involved in the direct regulation of dyneintubule sliding, or it may serve some coupled calcium transport function. At the concentration in which it is found, it must also at least act as a calcium buffer.

scholarly journals Glial hyaluronate-binding protein: a product of metalloproteinase digestion of versican?

1995 ◽  
Vol 312 (2) ◽  
pp. 377-384 ◽  
Author(s):  
G Perides ◽  
R A Asher ◽  
M W Lark ◽  
W S Lane ◽  
R A Robinson ◽  
...  
Keyword(s):  
Amino Acid ◽  
Human Brain ◽  
Peptide Mapping ◽  
Binding Protein ◽  
Protein A ◽  
Immunoblot Analysis ◽  
Bovine Brain ◽  
Naturally Occurring ◽  
Brain Extracts ◽  
The Brain

Glial hyaluronate-binding protein (GHAP) is a 60 kDa glycoprotein with an amino acid sequence identical to that of the hyaluronate-binding region of versican, a large fibroblast aggregating proteoglycan found in the brain. Both GHAP and versican were identified by immunoblot in bovine brain extracts prepared only minutes after death. Human recombinant collagenase, stromelysin, mouse gelatinase and gelatinases isolated from human brain by affinity chromatography digest versican and give rise to a polypeptide with electrophoretic mobility identical to GHAP. Immunoblot analysis, peptide mapping and C-terminal amino acid sequencing indicate that the polypeptide generated by digestion with human brain gelatinases is identical to GHAP. We suggest that GHAP is a naturally occurring versican degradation product.

scholarly journals Identification, purification and characterization of a membrane-associated N-myristoyltransferase inhibitor protein from bovine brain

1993 ◽  
Vol 291 (2) ◽  
pp. 635-639 ◽  
Author(s):  
M J King ◽  
R K Sharma
Keyword(s):  
Gel Filtration ◽  
Subcellular Fractionation ◽  
Bovine Brain ◽  
Particulate Fraction ◽  
Inhibitor Protein ◽  
Purification And Characterization ◽  
Sds Page ◽  
Protease Degradation ◽  
Myristoyl Coa

N-Myristoyl-CoA: protein N-myristoyltransferase (NMT) is the enzyme that catalyses the covalent transfer of myristic acid from myristoyl-CoA to the N-terminal glycine residue of a protein substrate. Subcellular fractionation of bovine brain indicates that NMT activity was located in both the cytosolic and the particulate fraction of the cell. Removal of the particulate fraction resulted in a 2-fold enhancement of NMT activity. Reconstitution of the particulate fraction and cytosolic fraction resulted in inhibition of the elevated cytosolic NMT activity. These results indicated the existence of putative inhibitor(s) activity of NMT located in the particulate fraction of bovine brain. The inhibitor was stable to heat and was identified as a protein, on the basis of its susceptibility to the proteases trypsin and chymotrypsin. Protease degradation first required the delipidation of the particulate fraction. The inhibitor was purified to near-homogeneity by heat treatment, solvent extraction and Sephacryl S-300 gelfiltration column chromatography. The inhibitor was purified 630-fold from the particulate fraction with a 20% yield. The protein inhibitor had an apparent molecular mass of 92 kDa by gel filtration and 71 kDa by SDS/PAGE, indicating the protein is monomeric. The inhibitor did not interact directly with myristoyl-CoA and possessed no protease, thioesterase or demyristoylase activity. Purified inhibitor protein inhibited the formation of 1167 pmol of myristoyl-peptide/min per mg of protein.

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